CELL CYCLE-DEPENDENT, INVITRO ASSEMBLY OF MICROTUBULES ONTO THE PERICENTRIOLAR MATERIAL OF HELA-CELLS

A centriolar complex comprising a pair of centrioles and a cloud of pericentriolar material is located at the point of convergence of the microtubules of the mitotic apparatus. The in vitro assembly of microtubules was observed onto these complexes in the 1,400 g supernatant fraction of colcemid-blocked, mitotic HeLa cells lysed into solutions containing tubulin and Triton X-100. Dark-field microscopy provided a convenient means by which this process could be visualized directly. When this 1,400 g supernate was incubated at 30°C and centrifuged into a discontinuous sucrose gradient, a band containing centriolar complexes and assembled microtubules was obtained at the 50-60% sucrose interface. Ultrastructural analysis indicated that the majority of the microtubules assembled predominantly from the pericentriolar material but also onto the centrioles. When cells were synchronized by a double thymidine block, the assembly of microtubules onto centriolar complexes was observed only in lysates of mitotic cells; no assembly was seen in lysed material of interphase cells. Microtubule assembly occurred onto centriolar complexes in solutions of either 100,000 g brain supernate, 2 x cycled tubulin, or purified tubulin dimers. This study demonstrates that the pericentriolar material becomes competent as a microtubule-organizing center (MTOC) at the time of mitosis. With use of the techniques described, a method for the isolation of centriolar complexes may be developed.